2004 Research Directory

JUNE 2004

This data was compiled in compliance with the New Jersey Commission on Spinal Cord Research's statutory mandate, N.J.S.A. 52:9E-1, “…to compile a directory of spinal cord research being conducted in the State.”

The information contained within this directory is not all-inclusive. The research projects and researchers listed in this directory are all based in the State of New Jersey, and have applied to and received funding during the fiscal year 2004 grant cycle. The research projects are not categorized, or listed in any particular order.

This directory is not a complete listing of all scientific research being performed within the State of New Jersey due to the proprietary nature of the research being conducted at various institutions throughout the State. In addition, institutions are not obligated to share their research information with the New Jersey Commission on Spinal Cord Research.

Please feel free to contact the New Jersey Commission on Spinal Cord Research at PO Box 360, Health & Agriculture Building, Market and Warren Streets, Trenton, New Jersey, 08625. The Commission's office can be reached by telephone at 609-292-4055, by fax at 609-943-4213, or by e-mail at NJCSCR@doh.state.nj.us.

The lack of substantial axonal regeneration in the adult mammalian central nervous system has been related primarily to the presence of molecules, which prevent regrowth of lesioned axons. Neutralization of these inhibitory activities would be one possibility to foster regrowth of severed axons. As a direct complementation to this approach we are proposing also to increase neurite outgrowth, enhance neuronal survival and allow remodeling of functional connections of denervated neurons in the central nervous system by introducing embryonic and neural stem cells that have been transfected to express the neural cell adhesion molecule L1 in membrane bound and soluble form into the lesioned spinal cord of adult mice. We have chosen L1 as a promising candidate for functional repair, since it has been shown to neutralize inhibitors present in the adult spinal cord, and to promote neurite elongation and axon regrowth on permissive mammalian Schwann cells by a homophilic binding mechanism between Schwann cells and neurons. Indicative of a beneficial effect of L1 is the observation that L1 is transiently upregulated by neurons and Schwann cells after a peripheral nerve lesion during nerve regeneration. Recently, L1 has also been shown to promote recovery of locomotion in a rat spinal cord lesion paradigm when applied transiently to the lesion site. These studies will now be extended by the use of genetically manipulated stem cells to allow severed axons and denervated neurons to restore functional connections by overcoming the largely inhibitory environment with the hope of eventually leading to spinal cord regeneration in higher vertebrates, and in particular, humans.

The applicant is an accomplished developmental neurobiologist who will use this award to develop and integrate a spinal cord injury program into his current research program. The applicant's current and past research emphasis has been on the proliferation of stem/progenitor cells in the developing brain and during adult neurogenesis, and he also is using genetic and genomic tools to identify the genetic regulators of stem/progenitor cell proliferation. This research project represents a new direction of research for the applicant who will bring his expertise in the area of the regulation of cell proliferation to the field of spinal cord injury. The project itself will focus on the important issue of the extent and role of cell proliferation after spinal cord injury. This is an appropriate focus because the applicant has developed many of the cell cycle methods that are used to assess cell proliferation and because the question of the role and extent of cell proliferation after spinal cord injury is an under-represented research area with many fundamental questions that are unanswered. The project will establish when, where, what kinds of cells proliferate, and what kinds of cells are produced. Then, the applicant will turn to mouse genetic resources to identify genetic differences in these traits and to identify the genetic loci that control these genetic differences. Finally, a series of experiments is proposed that will provide a bridge for using the special properties of proliferating cells as a therapeutic delivery system. At all stages, there will be interactions between the applicant's current stem/progenitor cell research program and the proposed project and, in addition, between the proposed project and the existing spinal cord research programs on the Robert Wood Johnson Medical School and Rutger's University campuses. The specific benefit of this project lies in its novelty and in the detailed methods that the applicant will apply to this issue. The fact that cell proliferation occurs after an injury has been known for a long time, but specific information about when, where and what kinds of cells are involved is simply not available, despite its importance in understanding the sequence of events occurring after the injury and during wound healing. This project will provide this information. In addition, the project has the goal of exploiting cell proliferation as a tool for the treatment and remediation of spinal cord injury. This is possible because proliferating cells have special biological properties, which make them a possible target for various treatments, ranging from available anti-mitotic drugs to genetically engineered retroviral vectors for the delivery of special purpose molecular treatments.

In today's society, the ability to drive an automobile is a fundamental skill that influences various aspects of daily living and can impact on individuals' level of independence. For many persons with spinal cord injury (SCI), the ability to drive is an indispensable means of returning to work, attending medical appointments, completing daily activities and participating in community events. Because of this, it is clear that the loss of the driving privilege can have a significant impact on the quality of life of individuals with SCI. In fact, studies have demonstrated that the inability to drive a car can significantly hinder an individual's return to employment (McShane & Karp, 1993), can result in specific aspects of community non-involvement (Shur & Kruse, 2000) and has been associated with emotional difficulties (Tachakra, 1981). Given these facts, it is important to develop interventions to help person's with SCI return to driving quickly and safely. The current research study will examine a new method for improving driver retraining for persons with SCI and subsequently improve overall quality of life. Specifically, through the application of an innovative technology, virtual reality (VR), the current proposal seeks to directly address current limitations in SCI driver re-training procedures and enhance current rehabilitation protocols for the restoration of driving capacity.

By developing a VR-based training protocol, subjects will be re-trained in "virtual driving environments". The study will examine whether the addition of this technology based training will result in a reduction in the total number of sessions required for driver re-training of SCI drivers learning to use adaptive equipment. Additionally, researchers will examine the influence of VR on self-rated measures of self-confidence of driving capacity and overall self-reported changes in quality of life, after returning to driving.

Finally, because there are only a handful of studies applying this promising new technology to clinical populations, the proposed study will attempt to identify any factors, positive or negative, to the use of this technology with SCI populations. To accomplish this objective, the study will compare performance of two groups of subjects: 25 SCI subjects receiving VR training sessions and 25 SCI subjects receiving traditional training sessions only. The VR retraining will consist of three VR sessions, each lasting approximately 1 hour, during which time subjects will be instructed to drive through two 20-minute VR driving environments, delivered via a modified driving console fitted with adaptive driving equipment. Measures of driving performance and self-efficacy ratings will be obtained for both groups during the evaluation and re-training protocol. One month after re-training, all subjects will be contacted via telephone, to inquire about driving status and complete quality of life questionnaires. It is anticipated that individuals receiving VR training, will require a fewer number of training sessions, and will have higher ratings of self-efficacy and quality of life.

Project TitleThe Development of Spinal Cord Therapies through a Genetic Analysis of Mouse Spinal Cord Development

The ultimate goal of spinal cord regeneration research is to incorporate new neurons into the adult CNS so that motor and sensory functions are restored. Stem cells provide a potential therapeutic route for treating spinal cord injury and disease. For these cell based therapies to work, stem cells must be instructed to a spinal cord lineage so they can differentiate into functional neurons. During development, a similar process occurs where neural progenitors are exposed to developmental signals and different types of spinal cord neurons are generated. Exposure of stem cells to developmentally important signaling molecules have led to the partial recovery of spinal cord function. These results are exciting and suggest that exposing stem cells to additional developmental factors could result in greater functional recovery.

By studying a mouse mutant called vacuolated lens, we have recently identified a new gene important for spinal cord development. The vacuolated lens mutation causes abnormal spinal cord development, which is exhibited by an over-production of spinal cord neurons and spina bifida (a failure to close the neural tube during development). Recently, we have identified the mutation responsible for these abnormalities. The mutation deletes a previously unidentified protein that enables cells to respond to their environment. The vl gene belongs to a family of protein called receptors that bind small proteins or chemicals (ligands). Thus, our research on the vacuolated lens mutant has identified a novel ligand-receptor signaling pathway that is required for normal spinal cord development.

Further research on the vacuolated lens mutation will benefit spinal cord research in two ways. First, the over-production of spinal cord neurons in mutant embryos suggests that the vacuolated lens pathway regulates the generation of spinal cord neurons during development. Thus, the further characterization of the vacuolated lens mutation will identify additional components of the pathway, which can be used in the future to create better stem cell therapies. For example, exposure of stem cells to the vacuolated lens ligand could result in more stem cells differentiating into functional spinal cord neurons, which might lead to greater functional recovery. Second, the vacuolated lens mutation also displays spina bifida, a developmental disorder that often results in paralysis. The further investigation of the vacuolated lens mutation will also lead to a greater understanding of this disease so that better preventative measures and/or treatments can be created.

Radial glial cells are neuronal stem cells that are found only transiently in the developing nervous system. They are the major source of new cells including neurons in the developing nervous system and provide guides for neurons to migrate to their appropriate destinations. We believe that these cells can be used to promote recovery following injury to the central nervous system. To overcome their innate instability in the mature brain, we have begun to analyze factors that can promote their persistence and infiltration into white matter of the mature spinal cord by preventing their differentiation. Radial glial cells have been found to form bridges across spinal cord lesions acutely following injuries in rats, inhibit formation of the glial scar, and promote behavioral recovery. Preliminary experiments suggest that they also improve recovery when transplanted chronically following spinal cord contusion in rats, possibly by providing growth and survival factors that are found in embryos.

The goal of this proposal is to understand cellular factors that can be used to stabilize radial glia and promote their persistence for longer periods of time in the adult spinal cord. Towards this goal, we have begun to identify factors that inhibit differentiation of radial glia. Radial glia will be modified to prolong their persistence in the spinal cord and then they will be transplanted into the spinal cord following contusion. The outcome measures in the experimental animals will include BBB scoring to analyze behavior and histology to analyze protection of secondary nerve damage and nerve regeneration. Our recent studies indicate that radial glial can promote recovery in the first 6 weeks following injury. The present studies will probe further whether this can be attributed to protection from secondary nerve damage. Longer-term studies for 12 weeks with tract tracing techniques will be used to analyze whether nerve regeneration is also promoted by the radial glia. Our recent studies also suggest that we can direct radial glial cells towards the phenotype of oligodendrocytes, which are the cells that produce myelin and provide vital insulation for nerves. Ultimately, the ability to turn radial glia into oligodendrocytes or to reintroduce oligodendrocytes may further improve recovery by providing myelination and more efficient impulse conduction along nerves. Therefore, we will determine whether transplanted radial glia show evidence of oligodendroglial differentiation following transplantation and we will consider factors that can promote this advantageous phenotype from radial glia.

Project TitleSCI Databases and Models: Accelerating Research with Information Technology

Our goal is to provide information technology (IT) tools to accelerate spinal cord research. We developed an open-source database named SCI-Base to manage all records from animals used in spinal cord injury research projects. We now plan to: 1) continue development of our SCI-Base animal database to extend reporting functions, add new data tables for new functional assays, and improve dependability and functionality, 2) work with Dr. Frank P.T. Hamers to integrate his computer program for automated scoring of the BBB locomotor function system into SCI-Base, and to add the new BMS (Basso Mouse Scale), and 3) work with Dr. Hamers to develop, validate, and integrate new software and hardware for the MASCIS device interface. The database and software we will develop will be used as a foundation for testing neuroprotective and regenerative therapies in animals prior to clinical trials. Each of our aims will enhance productivity and efficiency of hundreds of spinal cord research labs throughout the world.

Spinal cord injury is one of the most devastating neurological injuries affecting millions of people worldwide. Functional recovery after spinal cord injury requires regeneration and elongation of nerve fibers, followed by re-establishment of specific neuronal connections. However, in the adult central nervous system (CNS), regeneration and recovery of neuronal connections are limited. Among many issues, inhibitory/repulsive factors at the injury site present a major obstacle for the growth and re-wiring of regenerating axons. Tremendous efforts have been directed towards understanding the molecular and cellular mechanisms underlying axonal growth and inhibition in the CNS, with the hope that information derived from basic science research will provide the solid foundation required for developing strategies to overcome the growth inhibition by these molecules, thus leading to enhanced recovery. It is known that many of these axon inhibitory molecules elicit complex signal transduction pathways to exert their inhibition on the motility of regenerating axons. Therefore, elucidating the signal transduction mechanisms of inhibitory molecules is of particular importance as it can provide ways to disrupt signal transduction of these molecules, thereby attenuating their inhibitory effects and leading to growth promotion of regenerating axons. While numerous proteins, including membrane receptors and intracellular signaling molecules, have been intensively studied, other cell components can also contribute to axonal inhibition. The study proposed here would concentrate on the role of the special lipid structures on the cell membrane, the so-called lipid rafts. Lipid rafts are ordered membrane microdomains, enriched with cholesterol and glycosphingolipids, and are hypothesized to provide the signaling platforms for many external cues. However, whether lipid rafts play a role in axon guidance, inhibition, or nerve regeneration remains to be determined. Recent preliminary data from this lab provide direct evidence that lipid rafts mediate inhibitory signaling of several repulsive cues. The current hypothesis is that lipid rafts provide a common platform that is required for signal transduction of inhibitory molecules. Therefore, it is conceivable that manipulating lipid rafts would attenuate the signaling of inhibitory molecules to block inhibition, and subsequently lead to enhanced axonal elongation. These studies are expected not only to provide significant insights towards understanding the molecular and cellular mechanisms underlying axon inhibition, but they would also provide a foundation for developing innovative approaches to combat axon inhibition by targeting membrane lipids and other membrane components that constitute lipid raft microdomains.

The goal of this proposal is to attempt to repair spinal cord injuries by implanting into the lesion nanofibers whose surfaces are derivatized with bioactive peptides. These peptides, derived from sequences within the neuroregulatory molecule tenascin-C, have been demonstrated in vitro to increase axonal growth and regeneration. We have identified distinct peptides that either increase neurite growth in culture or provide directional cues to growing neurites, a function defined as neurite guidance. These peptides can overcome inhibition to neuronal growth caused by normally repulsive chondroitin sulfate proteoglycans, a major type of inhibitory molecule in the glial scar. This observation is highly significant because full recovery of function following CNS injury cannot occur unless axons a) elongate and b) are guided across the inhibitory terrain of the glial scar. Our goal is to evaluate whether the peptides can similarly overcome CSPG inhibition to axonal regeneration in an animal model. The peptides will first be chemically coupled to the surface of nanofibers that have been prepared by electrospinning a polymer solution of polycaprolactone (a biodegradable plastic) or nylon (an insoluble plastic). The resulting fibers have been demonstrated to be biocompatible and permit normal cell growth. The use of these modified fibers will thus provide us with the opportunity to sequester the neuroactive molecules within specific regions of the damaged spinal cord and in this manner provide: a) a scaffold for neuronal attachment and axonal outgrowth, b) an attachment site for the peptide that will prevent their diffusion from the site of injury, and c) a bridge across the glial scar. This will be the first use of nanofiber technology for the development of peptide-modified matrices for use in therapies designed to treat spinal cord injury.

There is mounting evidence that oxidation is a trigger of cell death and tissue damage in spinal cord injury. A potentially critical target of oxidation is the modification and inactivation of a transcription factor, HSF1, needed to mount a defensive response against stress. This defense mechanism involves the production of a family of molecular chaperones, the heat shock proteins (HSPs), that perform manufacturing support and quality control roles in protein homeostasis, ensuring that newly synthesised proteins are complete, taken to the correct position within the cell's structure and correctly folded, and where there is a problem, the HSP chaperones will also direct a non-functional protein for degradation. Indeed, the increased production of HSP chaperones provides a powerful survival mechanism in an acute situation, and this has been duly noted: "It provides good evidence that heat shock protein has its arms around neuroprotective levers in the cell."; while the technical obstacles of converting the finding into a therapy for stroke are formidable, "it's not beyond the pale."

Our working hypothesis is that oxidation, dysfunction of HSF1, and the decreased production of HSPs are sequential events that contribute to neuronal cell vulnerability in the injured spinal cord. We suggest that experimental means that can boost the chaperoning function in cells - either by the forced expression of redox modifiers to prevent/reverse the inactivation of HSF1 and enhance the production of HSP molecular chaperones, or by the use of chemical chaperones - would enhance the survivability of cells under adverse conditions.

We note that the ability to minimize cell death cord is critical to functional recovery after spinal cord injury as studies showed that survival of a mere 10 percent of the neurons could allow the patient to maintain significant capabilities. It is our sincere hope that this study of defining the function of molecular and chemical chaperones as neuroprotective mechanisms and if exploring means to boost this protective mechanism will contribute to the synergistic development of novel therapies to improve the survivability of neuronal cells under stress and, in so doing, preserve functionality and improve the long term outlook of spinal cord injured patients.

Project TitleDefining the Role of Necrosis Suppressor Calmyrin in Neuronal Death

Necrotic cell death, often initiated by ion channel hyper-activation, plays a major role in the initial and prolonged death of neurons consequent to spinal cord injury. Blocking or delaying such necrotic cell death would significantly limit the neuronal damage that is so incapacitating consequent to injury. A more detailed understanding of the molecular mechanisms of neuronal injury is required to design novel effective therapies. My project has the goal of extending understanding of molecular mechanisms of necrosis.

Our lab has been studying molecular mechanisms of necrotic neuronal death that occurs when ion channels are hyper-activated. Our approach toward this problem has been to use genetic strategies uniquely applied in the facile experimental model system C. elegans to identify genes required for necrosis under in vivo, physiological conditions. Our work to date indicates that neurotic cell death can be initiated by elevated activity of distinct mutant ion channels. As a consequence of elevated ion influx, there is a catastrophic elevation of intracellular calcium activates calpains (calcium-activated proteases) and other proteases to execute necrotic cell death. Importantly, this death mechanism appears highly conserved from nematodes to humans. For example, ion channel hyper-activation, ER calcium release and calpain activation are all mechanistic features of necrotic cell death that accompanies spinal cord injury.

One of the great advantages of the C. elegans model system is the capacity to use RNAi interference techniques to knock down the activities of defined genes. We have conducted a screen for necrosis suppressors in which neurons expressing hyper-activated ion channels are rescued from necrotic cell death by RNAi knockdown of specific genes. We have completed a screen of individual knockdown of all genes on chromosome 1 and we have identified 10 strong candidate necrosis suppressors. Of these death suppressors I am particularly interested in a homolog of human calmyrin--a protein that includes 3 EF hand calcium-binding domains and that has been previously implicated in mammalian cell death. I hypothesize that calmyrin performs a calcium-regulated function as a specific factor that executes necrosis and I suggest that as such, calmyrin would be a potential target for interventions that might prevent the effects of secondary necrosis that are so damaging in spinal cord injury. My plan is to test the main premise of my working hypothesis by defining how calmyrin fits into the necrosis pathway, deciphering how its knockdown suppresses necrosis and to begin to test roles for calmyrin in mammalian necrosis. My aims are: Aim 1 - to generate and perform basic characterization of a Ce-calmyrin knockout mutant. Aim 2 - to characterize the role of Ce-calmyrin in nematode cell death. Aim 3 - to test whether the human counterpart of Ce-calmyrin plays a conserved role in necrotic cell death.

This work will determine how Ce-calmyrin is required for necrosis, will position calmyrin activity in the genetic pathway for necrosis, will indicate how critical calmyrin calcium-binding capacities are for function, and will suggest whether calmyrin over-expression confers toxicity on its own exerts dominant-negative effects. I will also determine whether human calmyrin can substitute for the work gene and I will seek evidence that mammalian calmyrin influences necrotic responses in cultured mammalian neurons. The ultimate outcome of this work should be significant because I will define a component of the molecular mechanism operative in necrosis and it will provide a molecular description of one way in which necrotic cell death can be suppressed in a physiological context.

The devastating consequences of spinal cord injury are largely attributed to death of neurons destroyed by the mechanical crush and the progressive necrosis of neurons subjected to the release of the toxic molecules from initially damaged neurons. It is clear that preventing injury-induced secondary necrosis is a critical intervention goal, since necrosis is a profound contributor to neuropathology in spinal cord injury. Identifying the underlying molecular mechanisms of necrosis and defining ways to block necrosis may therefore inspire novel treatments for SCI.

Our lab is using the unique genetic and molecular biology tools developed in the facile and powerful model organism Caenorhabditis elegans to elaborate the molecular mechanisms of necrosis by identifying the genes that are critical for necrosis regulation and execution. Since most basic biological processes, including cell death, are conserved from nematodes to humans, key players in necrosis that are identified in the simple nematode C. elegans model are highly likely to be relevant to advancing understanding of the molecular mechanisms of spinal cord injury.

In spinal cord injury, loss of Na+ homeostasis is clearly an early critical event both in secondary neural necrosis and in secondary axonal damage. The elevation of intracellular Ca+2 after collapse of Na+ gradient activates calcium-dependent proteases, such as calpain, resulting neuron degeneration. We have developed a C. elegans model in which a mutant sodium channel is hyper-activated, inducing necrosis dependent on elevation of intracellular calcium. Like apoptotic cell death mechanisms, necrotic cell death mechanisms are highly conserved between nematodes and humans. A key advantage of the C. elegans model is that one can conduct genetic screens for mutations that affect a process of interest that any preconceived notion of what that might be. I will begin with a strain that includes a weak genetic inducer of necrosis and I will screen for novel secondary mutations that enhance death. The normal function of such genes is to protect against necrosis in a physiological context-genes of great interest.

Aim 1 is to conduct a screen of the C. elegans genome to identify mutations that enhance progression through necrosis, the normal function of which could be neuroprotective. The plan is to begin with a strain in which specific labeled neurons undergo inefficient necrosis induced by Na+ channel hyperactivation-neurons will fluoresce green if alive. After mutatgenesis, I will look for rare strains in which necrosis is enhanced and green signals disappear or are significantly diminished.

Aim 2 is to perform tests that characterize enhancer mutants and prioritize most promising loci for molecular study. I will perform basic characterization of death-enhancer mutants to identify novel genes that protect against necrosis. Then I will position those genes actions in the necrosis pathway. Finally, I will precisely map the chromosal locations of the top priority genes to facilitate their future molecular analysis.

Currently, there is no efficacious treatment that limits the necrotic-like cell death that accompanies SCI and makes a major contribution to disability. Powerful genetic approaches not readily applied in other systems can provide novel insights into basic mechanisms of necrotic cell death. My work should identify currently known components of this pathway that normally help protect the neuron from proceeding into necrotic cell death. Information generated will advance the elaboration of molecular mechanisms of injury-induced neuronal cell death and might ultimately suggest novel strategies for therapies in SCI.

Our research is focused on studying the development of the central nervous system. Specifically, we are interested in the developmental role played by closure of the neural tube, the structure that gives rise to the brain and spinal cord. Spina bifida, or failure of the neural tube to close, is the model we are using to study the role of neural tube closure. Spina bifida is a common human birth defect, and occurs in one out of every 1000 live births (Harris & Juriloff, Hum Mol Gen 2000, 9(6):993-1000). Spina bifida in humans is characterized by the overgrowth of neural tissue in the open spinal cord, and this overgrowth leads to incontinence and paralysis of the lower limbs (Northrup & Volcik, Curr Probl Pediatr 2000, 30(10):313-32). To study spina bifida, we are using the mouse mutant Splotch-delayed, which is a spontaneous mutation within the gene Pax 3 that causes a spina bifida associated overgrowth very similar to the human form of the defect. The goal of the present research project is to determine what role neural tube closure plays in regulating this overgrowth.

This research will benefit spinal cord injury research in two ways. First, the study of how neural tube closure acts to regulate proliferation and differentiation in the spinal cord will directly contribute to our ability to manipulate stem cells for spinal cord transplant. Second, understanding how spina bifida leads to paralysis through the disrupted regulation of proliferation and differentiation will enhance our understanding of spinal cord injury associated paralysis and our ability to treat paralysis.

Preliminary data indicates that once the neural tube fails to close, the normal processes of proliferation and differentiation are perturbed, resulting in a spinal cord that is approximately one and a half times the size of the normal neural tube. We have quantified this increase in a subset of proliferating cells and mature differentiated cells, through analysis of proliferative cells and many subtypes of spinal cord motor neurons and interneurons using cell-specific proteins. Further analysis of additional populations will determine whether there is a general increase in populations of neurons making up the spinal cord.

We can determine whether failure of neural tube closure is directly responsible for the overgrowth phenotype by allowing the neural tube to close in Splotch-delayed animals. Experimentally, this can be accomplished via supplementation with folic acid, a B vitamin known to decrease the incidence of spina bifida (Fleming & Copp, Science 1998, 280(5372):2107-9). The same neural tube cell type analysis discussed above will determine the effect of closing the neural tube in Splotch-delayed animals on cell proliferation and differentiation.

Spinal cord injuries (SCI) result in loss of neurons and as a consequence, a disruption of the circuits to which these cells contribute, thereby resulting in sensory and locomotor deficits. Therapies for SCI aim to restore these circuits for which they have to first regenerate the lost neurons. The spinal cord however, is made up of multiple neuronal classes (broadly classified as sensory neurons, motor neurons and interneurons) that have distinct and specific roles in wiring the neuronal circuits. It is critical to keep this in mind while trying to encourage endogenous "stem cells" or transplanted pleuripotent cells to differentiate into functionally distinct neuronal sub-types, which can then re-establish functional networks. To do so the mechanisms of early neuronal development that result in generation of distinct neurons from common progenitors need to be critically understood and followed.

The developing spinal cord specifies multiple sub-types of neurons from common "stem cell" like progenitors. Expression of proteins known as transcription factors in the progenitors leads to the specification of neuronal sub-types. As a result, distinct cell types are defined at distinct positions at specific levels of spinal cord. These patterning cues result in placing sensory neurons that relay messages from the skin to the spinal cord at one end (dorsal) and motor neurons at the other (ventral). Alterations in expression patterns of progenitors leads to miss-specified cell fates that form aberrant neuronal circuits. Pax6 is one such patterning gene that is expressed broadly in precursors of all three neuron types and is known to alter cell fates in the developing spinal cord. However, while numerous studies have characterized the role of Pax6 in determining neuronal identity in the spinal cord, the mechanisms that determine neuronal fates downstream of this factor are poorly understood. Our analysis of the spinal cord of mutant mice having no functional Pax6 has shown a mis-specification of dorsal neurons in the ventral spinal cord. This suggests a role for Pax6 in controlling broad D/V neuronal identities and we hypothesize that Pax6 controls the expression of factors that are in part responsible for distinguishing dorsal from ventral progenitor cells. The studies outlined in this proposal will address the poorly understood molecular mechanisms of Pax6 function in determining the fate of progenitors and will be directly relevant to clinical efforts to manipulate functional neuronal circuitry following spinal cord injury.

Rutgers, The State University of NJ Department of Biomedical Engineering 617 Bowser Road Piscataway, New Jersey 08854-8014 732-445-3722

Project TitleIn Vivo Tissue-Level Thresholds for Spinal Cord Injury

New Jersey currently has approximately 6,000 residents with spinal cord injuries with about 300 new injuries occurring each year. The effects of this debilitating disease create an enormous personal toll on the individuals and families. Thus, the overall long-term objective of this research is to identify the conditions that cause spinal cord injury (SCI) in humans and prevent new injuries from occurring.

Injury to the spine frequently results in a dynamic, compressive load to the spinal cord. One of the manifestations of this compressive load is injury to the microvasculature -- demonstrated as gross hemorrhage and permeability changes in the blood spinal cord barrier -- that contribute to the devastating secondary insults that dictate the overall neuropathology. Therefore, prevention of vascular injury would limit many of the devastating effects of SCI. However, the tolerance of spinal cord tissue to compressive loads is unknown, even though models of spinal cord injury, such as the Impactor contusion model, are commonly used to produce microvascular, contusion injuries. In the Impactor model, a weight is dropped from a predefined height on to the exposed spinal cord of the rat, under extremely well controlled conditions. We will characterize the severity and extent of microvascular injury following contusion initiated with the Impactor model. We will simultaneously develop a computer model of the Impactor that allows us to understand the stress and strain in the spinal cord that result from weight drop. By statistically comparing the results from the experimental Impactor studies to the computer model, we will identify the mechanical tolerance of spinal cord vasculature. These results can be combined with models of spinal trauma to distinguish how different mechanisms of vertebral damage result in specific injuries to the spinal cord vasculature, and therefore are invaluable in developing means to prevent SCI in humans. Moreover, the results can be used in combination with simulations of SCI with other experimental methods or locations to predict patterns of SCI, and therefore optimize experimental trauma systems to study specific injuries. Following completion of this research project, we will have identified the threshold values of mechanical variables that best predict microvascular injury following SCI and could possibly prevent and save some of the enormous consequences of the 300 new injuries that occur each year.

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